The aversive aspects of withdrawal from chronic nicotine exposure are thought to be an important motivational factor contributing to the maintenance of the tobacco habit in human smokers. Much emphasis has been placed on delineating the underlying neurobiological mechanisms mediating different components of the nicotine withdrawal syndrome. Recent studies have shown that both central and peripheral populations of nicotinic acetylcholine receptors (nAChRs) are involved in mediating somatic signs of nicotine withdrawal as measured by the rodent nicotine abstinence scale. However, only central populations of nAChRs are involved in mediating affective aspects of nicotine withdrawal, as measured by elevations in brain-stimulation reward thresholds and conditioned place aversion. Nicotine interacts with several neurotransmitter systems, including acetylcholine, dopamine, opioid peptides, serotonin, and glutamate systems. Evidence so far suggests that these neurotransmitters play a role in nicotine dependence and withdrawal processes. The available evidence also suggests that different underlying neurochemical deficits mediate somatic and affective components of nicotine withdrawal. The aim of the present review is to discuss preclinical findings concerning the neuroanatomical and neurochemical substrates involved in these different aspects of nicotine withdrawal.